Epidermal Growth Factor Receptor (EGFR) is a specific receptor for epidermal growth factor (EGF) and transforming growth factor-.alpha. (TGF-.alpha.). When these mitogenic polypeptides bind to EGFR, tyrosine kinase activity of the receptor is induced, and this in turn triggers a series of events which regulate cell growth. A number of malignant and non-malignant disease conditions are now believed to be associated with EGFR, particularly aberrant expression of EGFR. Aberrant expression includes both increased expression of normal EGFR and expression of mutant EGFR. Overexpression of EGFR is found in any human tumors including most glioblastomas and breast, lung, ovarian, colorectal, bladder, pancreatic, squamous cell and renal carcinomas. Elevated EGFR levels correlate with poor prognosis in human tumors. EGFR is also implicated in nonmalignant diseases, such as psoriasis. The sequence of the mRNA encoding human EGFR is known. Ullrich et al., Nature, 1984, 309, 418; GenBank Accession Number X00588. The gene encoding EGFR is also known as c-erb-B1. Two EGFR transcripts typically appear on Northern blots, one measuring 10 kb and one measuring 5.6 kb.
A number of inhibitors of EGFR have been shown to be effective in inhibiting the growth of human tumor cells. Monoclonal antibodies to EGFR and drugs which inhibit EGFR tyrosine kinase activity can inhibit the growth of human cancer cell xenografts in nude mice. Normanno et al., Clin. Cancer Res., 1996, 2, 601. The drug PD153035, which inhibits EGFR tyrosine kinase activity, can inhibit the growth of A431 cells in nude mice, and tyrphostins, which inhibit the activity of EGFR as well as other tyrosine kinases, have been shown to inhibit the growth of squamous carcinoma in nude mice. Kunkel et al., Invest. New Drugs, 1996, 13, 295 and Yoneda et al., Cancer Res., 1991, 51, 4430.
Vectors expressing EGFR nucleic acid sequences in an orientation complementary to mRNA have been used to study the effects of EGFR on proliferation of cultured cancer cells. Transfectants of the human epidermoid carcinoma KB cell line expressing EGFR cDNA or RNA sequences in an orientation complementary to mRNA exhibited restored serum-dependent growth and impaired colony formation and growth in agar. Moroni et al., J. Biol. Chem., 1992, 267, 2714. Human pancreatic carcinoma cells of the PC-7 cell line transfected with vectors expressing EGFR cDNA sequences in an orientation complimentary to mRNA showed inhibited cell growth, colony formation and tumorigenicity in nude mice. Liu et al., Chinese Medical Journal, 1995, 108, 653. Transfection of human colon cancer cell lines with EGFR RNA expression vectors producing an oligonucleotide complementary to mRNA caused a reduction in cell proliferation and ability to grow on soft agar. Rajagopal et al., Int. J. Cancer, 1995, 62, 661. Human rhabdomyosarcoma cells transfected with a plasmid expressing EGFR cDNA in an orientation complementary to mRNA had greatly impaired proliferation. De Giovanni et al., Cancer Res., 1996, 56, 3898.
Considerable research is being directed to the application of oligonucleotides complementary to mRNA and other oligomers for therapeutic purposes. Oligonucleotides complementary to mRNA have already been employed as therapeutic moieties in the treatment of disease states in animals and man, and compositions comprising oligomers complementary to mRNA have been shown to be capable of modulating expression of genes implicated in viral, fungal and metabolic diseases. Further, oligonucleotides complementary to mRNA have been safely administered to humans and clinical trials of approximately a dozen oligonucleotide drugs targeted to viral and cellular gene products are underway.
Oligodeoxyribonucleotides complementary to mRNA targeted to EGFR have been encapsulated into liposomes linked to folate via a polyethylene glycol linker and delivered into cultured human epidermoid carcinoma KB cells. The oligonucleotides were a phosphodiester (P=O) 15-mer complementary to the EGFR gene stop codon, or the same sequence with three phosphorothioate (P=S) linkages at each end. Both of these oligonucleotides reduced KB cell proliferation by greater than 90% after treatment with 3 .mu.M oligonucleotide in folate-PEG-liposomes. In contrast, free P=O oligonucleotide caused almost no growth inhibition, and free P=S-capped oligonucleotide caused only a 15% growth inhibition, even at this high dosage level. EGFR expression, measured by indirect immunofluorescence, was virtually abolished in cells treated with either of the folate-PEG-liposome-encapsulated oligonucleotides but EGFR expression was qualitatively similar to untreated cells after treatment with free oligonucleotide. Wang et al., Proc. Natl. Acad. Sci. U.S.A., 1995, 92, 3318.
A 15-mer phosphorothioate oligonucleotide complementary to the translation initiation region of EGFR mRNA was found to inhibit cell proliferation by over 25% in A431 cells, derived from a vulval carcinoma. This activity, though dose-dependent from 1-25 .mu.M, was not mediated by an antisense mechanism, as demonstrated by a lack of reduction in either EGFR protein or mRNA after oligonucleotide treatment. In addition, an 18-mer oligonucleotide complementary to mRNA targeted to the same region had no effect even at the highest (25 .mu.M) dose, and neither oligonucleotide had any effect in the two other tumor-derived cell lines tested. Coulson et al., Mol. Pharm., 1996, 50, 314.
The suppression of growth of pancreatic carcinoma cell lines by undisclosed oligonucleotides complementary to mRNA inhibiting the expression of TGF-.alpha. and/or the EGFR has been reported. Hall et al., unpublished data, reported in Hall and Lemoine, Models of Pancreatic Cancer, in Cancer Surveys, Volume 16: The Molecular Pathology of Cancer, 1993, p.135-155.
Rubenstein et al. have reported treatment of established human-derived prostate tumor xenografts in nude mice by intralesional injection of oligonucleotides complementary to mRNA directed against mRNAs encoding TGF-.alpha. and EGFR. The oligonucleotides included 39-mers complementary to 18 bases located 5' and 3' from the AUG mRNA translation initiation codon of either TGF-.alpha. or EGFR sequence. The oligonucleotides were phosphorothioated at each of three terminal bases at both the 5' and 3' ends. The oligonucleotides were administered either alone or in combination, with the combination treatment proving most effective. J. Surg. Oncol., 1996, 62, 194. In U.S. Pat. No. 5,610,288, Rubenstein et al. disclose polynucleotides of about 20 to 50 nucleic acid bases, most preferably about 40 nucleic acid bases in length, which preferentially hybridize to the start codon of the mRNA encoding EGFR. A preferred embodiment is a 39-mer including 18 bases complementary to the 5' side of the translation initiation codon. This oligonucleotide inhibited PC-3 cell growth when administered in combination with an oligonucleotide complementary to mRNA targeted to TGF-.alpha.. Alone, the EGFR oligonucleotide gave inhibition of cell growth equivalent to that achieved with an inverted (5' to 3') version of the same sequence.
Rearrangements or deletions of the EGFR gene resulting in mutant EGFR protein have been found in some cancers. The in-frame deletion from nucleotides 275-1075 in the EGFR has been referred to as class I, Type I or Type III mutation. WO 96/16988 (Wong et al.) discloses cell lines capable of overexpressing Type III mutant EGFR, vaccines for inhibiting tumor formation comprising peptides similar to a fusion junction present in mutant human EGFR, antibodies raised against a cell line overexpressing Type III mutant EGFR, and oligonucleotides complementary to mRNA targeted to a Type III mutant EGFR which decrease expression of a mutant EGFR. In a preferred embodiment, the oligonucleotide complementary to mRNA contains sequences from what were formerly distant portions of the normal EGFR cDNA. The oligonucleotide must contain the sequence 5'-TACCTT-3'. An 18-mer oligonucleotide containing this sequence was found to downregulate mutant EGFR levels when given at a 40 .mu.M dose in cultured cells which overexpressed Type III mutant EGFR.
The present invention provides new oligonucleotide compounds complementary to mRNA, as well as other oligonucleotide compounds, and compositions comprising the same together with methodologies for the use of certain compounds of the invention for interfering with translation of selected mRNA targets related to epidermal growth factor receptor.